MECHANISM OF ALLOSTERIC INFLUENCE ON ENZYME ACTIVITY

这一应用的长期目标是理解分子 变构配体改变酶行为的机制。 具体地说，猪心富马酸酶E.Coli的变构性质 氨基甲酰磷酸合成酶，牛心NAD依赖的异柠檬酸 脱氢酶和大鼠肝脏磷酸果糖激酶将被研究。 富马酸酶和氨基甲酰磷酸合成酶分别受 影响最大速度和/或米氏的变构修饰剂 通过改变蛋白质的三级结构来获得常数。动能 这些酶的行为将用热力学方法研究。 关联函数法。这一方法提供了对 修饰配体与游离酶的离解常数 修饰配体对最大速度的部分影响，以及 底物与变构相互作用的自由能 莱兰德。后两个参数描述了 变构配体一旦结合。通过观察这些参数的变化 通过系统地改变变构配体的结构而产生的 或蛋白质，我们将能够推断配体和蛋白质的结构特征 对变构配体作用的这两个不同方面很重要的蛋白质。 这一方法将被扩展到研究丹参的作用机制。 还可以改变酶的聚集状态的变构配体以及 将应用于异柠檬酸脱氢酶调节的研究 ADP和磷酸果糖激酶被多种调节配体所激活。 异柠檬酸脱氢酶应该提供一个简单的例子 变构作用的三级和四级之间的关系， 而磷酸果糖激酶是一个复杂的例子， 不同的相互依存的调控机制。尽管有这种复杂性， 应该通过定量评估 相关的自由能耦合参数。这种洞察力已经被遮蔽了 在过去，人们普遍认为这种酶可以存在于 只有两种可能的构象状态。这种新的见解尤其是 重要的是考虑到磷酸果糖激酶在 碳水化合物动态平衡。 这些研究所需的实验方法主要包括 酶动力学和荧光偏振测量，后者 在共价标记有合适外源物质的酶上进行 荧光探头。

The long term objective of this application is to understand the molecular mechanisms by which allosteric ligands modify enzymatic behavior. Specifically, the allosteric properties of porcine heart fumarase, E. coli carbamoyl phosphate synthetase, bovine heart NAD-dependent isocitrate dehydrogenase, and rat liver phosphofructokinase will be investigated. Fumarase and carbamoyl phosphate synthetase are each regulated by allosteric modifiers that influence maximal velocity and/or Michaelis constants by altering the tertiary structure of the protein. The kinetic behavior of these enzymes will be studied using a thermodynamic linked-function approach. This approach provides for the quantitation of the dissociation constant of modifying ligand from free enzyme, the fractional influence that the modifying ligand has on maximal velocity, and the free energy of interaction between the substrate and allosteric ligand. These latter two parameters describe the efficacy of the allosteric ligand once bound. By observing the changes in these parameters produced by systematically altering the structure of the allosteric ligand or protein, we will be able to infer structural features of the ligand and protein important for these two separate facets of allosteric ligand action. This approach will be extended to investigate the mechanism of action of allosteric ligands that alter an enzyme's aggregation state as well and will be applied to a study of the regulation of isocitrate dehydrogenase by ADP, and phosphofructokinase by a wide variety of regulatory ligands. Isocitrate dehydrogenase should provide a straightforward example of the relationship between tertiary and quaternary levels of allosteric action, whereas phosphofructokinase represents a complex example of several different interdependent regulatory mechanisms. Despite this complexity, significant new insight should be obtained by quantitatively evaluating the pertinent free energy coupling parameters. This insight has been obscured in the past by the common assumption that the enzyme can exist in one of only two possible conformational states. This new insight is especially important in view of the vital role phosphofructokinase plays in carbohydrate homeostasis. The experimental methodologies required for these studies consist primarily of enzyme kinetics and fluorescence polarization measurements, the latter being performed on enzymes covalently labeled with a suitable extrinsic fluorescent probe.